Chapter 3. Antigens Terminology: Hapten - a small molecule that is antigenic but not (by itself) immunogenic. Antigen : Substances that can be recognized by the surface antibody (B cells) or by the TCR when associated with MHC molecules Antibodies can be made to haptens only after the Immunogenicity VS Antigenicity: hapten is covalently conjugated to a large protein Immunogenicity – ability to induce an antibody and/or “carrier”. cell-mediated immune response Antigenicity – ability to combine with the final products of the response (antibodies and/or T cell receptor)
NOTE: Most immunogenic molecules are also antigenic
Figure 5.1
1 Factors that influence immunogenicity:
- Foreign-ness – non-self (far apart evolutionary) 2 - Type of molecule (chemical nature) - protein > polysaccharide > lipid > nucleic acid - Size - larger molecules tend to be more immunogenic 3 -Composition - heterogeneity increases immunogenicity. - 4ry > 3ry > 2ry > 1ry structure
-Degradability - protein antigens must be degraded (phagocytosis) in order to be presented to helper T cells .
- Physical Form - Denatured > Native
Additional factors that influence the immune response:
- Genetics of the recipient (genotype - MHC)
- Dosage of the antigen (optimal dose - tolerance)
- Number of doses of the antigen (boosters)
- Route of administration of the antigen - intravenous (spleen) - subcutaneous (lymph nodes) - intraperitoneal (lymph nodes) - oral (mucosal) - inhaled (mucosal)
- Use of adjuvant
1 Commonly used adjuvants: (Table 3.3)
Adjuvant : a substance that, when mixed with an antigen Alum - aluminum potassium sulfate - precipitates the antigen, and injected with it, serves to enhance the immune resulting in increased persistence of the antigen. Mild response to the antigen. granuloma.
Possible mechanisms of action of adjuvants : Incomplete Freund’s adjuvant - mineral oil-based - increases - Prolong the persistence of the antigen , thus giving the persistence of the antigen, mild granuloma, and induces co- immune system more time to respond stimulatory signals.
- Increase the “size” of the antigen by causing aggregation , Complete Freund’s Adjuvant - mineral oil-based adjuvant containing dead Mycobacterium - increases persistence of - Stimulate lymphocyte proliferation and/or activation the antigen, stimulates a chronic inflammatory response - Stimulate a local inflammatory response , thus recruiting (granuloma ), and co-stimulatory signals. Activates cells to the site of the antigen (GRANULOMA) Macrophages and DCs.
- Enhance co-stimulatory signals Bacterial Lipopolysaccharides - stimulate nonspecific lymphocyte activation and proliferation, and co- stimulatory signals.
Epitope or Antigenic Determinant - the region INCREASED COMPLEXITY of an antigen that binds to a T cell receptor or a B cell receptor (antibody).
- Since an epitope is the part of the antigen that binds to the B cell or T cell antigen receptor, it is the part that determines the antigenicity of the antigen - thus the term “antigenic determinant ”.
-T and B cells recognize different epitopes on an - Each different protein and glycoprotein of a virus (or bacterium antigen or foreign cell) constitutes a different antigen - Each different antigen contains a number of different epitopes
Properties of B cell epitopes (Table 3-4) Denaturation!!!!
- Usually dependent on the native, tertiary conformation of the antigen (PROTEIN FOLDING) PROTEIN - Must be accessible - tend to be on the “surface” of the FOLDING antigen (hydrophilic)
- May be made of sequential or non-sequential amino acid sequences (epitopes made up of non-sequential amino acid sequences are called “conformational epitopes”).
- Binds to soluble antigen, No MHC molecule requirement
- Large antigens contain multiple, overlapping B cell epitopes.
2 Properties of B cell epitopes (Table 3-4) HYDROPHILIC - Usually dependent on the native, tertiary conformation of the antigen
- Must be accessible - tend to be on the “surface” of the antigen (hydrophilic)
- May be made of sequential or non-sequential amino acid sequences (epitopes made up of non-sequential amino acid sequences are called “conformational epitopes”).
- Binds to soluble antigen, No MHC molecule requirement
- Large antigens contain multiple, overlapping B cell epitopes.
Properties of B cell epitopes (Table 3-4)
- Usually dependent on the native, tertiary conformation of the antigen
- Must be accessible - tend to be on the “surface” of the Anti-lysozyme (-) antigen (hydrophilic) Anti-lysozyme (+) - May be made of sequential or non-sequential amino acid sequences (epitopes made up of non-sequential amino acid sequences are called “conformational epitopes ”). Anti-lysozyme (+) - Binds to soluble antigen, No MHC molecule requirement
- Large antigens contain multiple, overlapping B cell epitopes.
Properties of B cell epitopes (Table 3-4)
- Usually dependent on the native, tertiary conformation of the antigen
- Must be accessible - tend to be on the “surface” of the antigen (hydrophilic)
- May be made of sequential or non-sequential amino acid A sequences (epitopes made up of non-sequential amino acid sequences are called “conformational epitopes”). B ** - Binds to soluble antigen, No MHC molecule requirement
1. Antibody binding may be lost after a protein is denatured!! - Large antigens contain multiple, overlapping B cell 2. ??? epitopes.
3 Properties of B cell epitopes (Table 3-4)
- Usually dependent on the native, tertiary conformation of the antigen
- Must be accessible - tend to be on the “surface” of the antigen (hydrophilic)
- May be made of sequential or non-sequential amino acid sequences (epitopes made up of non-sequential amino acid sequences are called “conformational epitopes”). "B-lymphocytes have sIg molecules on their surface that recognize - Binds to soluble antigen, No MHC molecule epitopes directly on antigens. Different B-lymphocytes are requirement programmed to produce different molecules of sIg, each specific for a unique epitope." - Large antigens contain multiple, overlapping B cell epitopes. animation and pictures from http://www.cat.cc.md.us/courses/bio141/lecguide/unit3/epsig.html
Large antigens contain multiple, Properties of T cell epitopes (Table 3-4) overlapping B cell epitopes. - Involves a tertiary complex: T cell receptor, antigen, and MHC molecule
Amino acids 1-12 Epitope 1 - Must be accessible - tend to be on the “surface” of the Amino acids 8-20 Epitope 2 antigen (hydrophilic) Amino acids 19-33 Epitope 3 - May be made of sequential or non-sequential amino acid sequences (epitopes made up of non-sequential amino acid sequences are called “conformational 1 10 20 30 110 epitopes”). Linear or Sequential Antigen - Binds to soluble antigen, No MHC molecule requirement
Would this cause cross-reactivity? - Large antigens contain multiple, overlapping B cell epitopes.
Properties of T cell epitopes (Table 3-4)
- Involves a tertiary complex: T cell receptor, antigen, and MHC molecule 3 - Internal linear peptides ( hydrophobic ) produced by 4 processing and bound to MHC molecules 2 - Binds to soluble antigen, No MHC molecule requirement
1 - Large antigens contain multiple, overlapping B cell epitopes.
4 Properties of T cell epitopes (Table 3-4)
- Involves a tertiary complex: T cell receptor, antigen, and MHC molecule
- Internal linear peptides (hydrophobic) produced by processing and bound to MHC molecules
- Does not bind to soluble antigen, APC processing
- Recognize mostly proteins but some lipids and glycolipids can be presented on MHC-like molecules
Properties of T cell epitopes (Table 3-4)
- Involves a tertiary complex: T cell receptor, antigen, and MHC molecule
- Internal linear peptides (hydrophobic) produced by processing and bound to MHC molecules
- Does not bind to soluble antigen, APC processing
- Recognize mostly proteins but some lipids and glycolipids can be presented on MHC-like molecules (remember CD1 molecules!) Must be processed & presented with MHC in APC!!!!
Immunoglobulin Structure and Function
5 1 - Each heavy and light chain 2 is made up of a number of domains (= Ig folding or Ig domains ). * * - Light chains consist of 2 domains ( C and V ). - Heavy chains have 4-5 domains (depending on * the class of antibody) - Each domain is about 110 Heavy chain= 446 aa Light chain= 214aa amino acids in length and 1 2 contains an intrachain disulfide bond between Constant Regions two cysteines about 60 amino acids apart. Variable Regions
-150,000 molecular weight Basic Antibody Structure • Multiple myeloma = cancerous plasma cells • Monomer = 150,000
1
2 3 4
What is the difference?
- Constant (C) and Variable (V) regions
RECAP: - The Fc region plays NO role in antigen binding. 100,000 MW 3 (Fab) 2 - Papain breaks antigen molecules into 2 Fab fragments and an Fc fragment.
- Pepsin breaks antibody molecules into an F(ab’)2 fragment and a VERY SMALL pFc’ fragment. 1 2 - Mercaptoethanol treatment results in 2 heavy and 2 2 (50,0000) light chains 2 (25,000) 2 H + 2 L - Complexes of antibodies cross-linked by antigen are Papain 1 called “immune complexes”. 2 (45,000) 1 (50,000) 2 Fab + Fc
6 1. Constant region - amino Summary acid sequence in the C- terminal regions of the H and • Molecule consists of Constant and Variable regions for 1 L chains is the same . both Light and Heavy chains (C H, V H, C L, V L) 2 • Ig molecule made of domains 2. Variable region - amino 1 • Domains ~ 110 aa 3 acid sequence in the N- terminal regions of the H and • Each antigen-binding site is made up of the N-terminal L chains is different . This domain of the heavy and the light chains region provides antibodies • IgM and IgE possess 4 C H domains (C H1-CH4). Hinge with unique specificity. region is missing. • IgG, IgA and IgD have 3 C H domains (C H1-CH3). 3. Hyper-variable regions are regions within the variable • Hypervariable regions in the Variable regions of both regions (greater H and L chains. specificities).
Figure 3.3
-Within the variable Antibody-Binding Site domains are three regions of extreme Complementarity-Determining Regions , or CDRs. variability.
These are referred to as the hypervariable regions.
These regions of the variable domains actually contact the antigen.
They therefore make up the antigen-binding site.
These regions are also called the complementarity- Heavy Chain Light Chain determining regions , or CDRs.
H chain CDRs - A simulated antigen- RECAP: binding site showing - Antibodies are comprised of repeating 110 aa units referred to how the CDRs form as domains or Ig folds. points of contact with - The C-terminal domains are constant from antibody to the antigen. antibody (within a class).
- The constant region domains are responsible for all functions L chain CDRs of antibody other than antigen binding ( opsonization, ADCC, complement activation) Biological Function!
- The N-terminal domains are variable from antibody to antibody and are referred to as “variable domains ”.
- The variable domains contain 3 hypervariable regions - the CDRs .
- The CDRs of the V domains in both H and L chains make up the antigen-binding site .
7 Fc γ receptors enhance phagocytosis of foreign cells/particles Antibody-Mediated Effector coated with IgG
Functions Antibody made in response to foreign cells (cells/viral • Binding to Antigen particles/bacteria etc) will bind to those cells. • OPSONIZATION: FcR in Macrophages and Macrophages (and neutrophils) possess receptors for the Fc neutrophils (C3b) region of IgG. • COMPLEMENT ACTIVATION: IgG and IgM Binding of macrophage Fc receptors to antibody bound to cells/particles facilitates and increases phagocytosis of • ADCC – NK cells trough FcR cells/particles. • CROSSING EPITHELIAL LAYERS – IgA (but also IgM) • CROSSING PLACENTA- IgG
ADCC - Antibody-dependent cellular cytotoxicity - mediated by IgG
Antibody made in response to foreign cells (cells/viral particles/bacteria etc) will bind to those cells.
Cells of the innate immune system (neutrophils, eosinophils, macrophages, NK cells) possess receptors for the Fc region of IgG.
These cells bind to antibody on the surface of foreign cells Monomer, and release lytic compounds Dimer, lysis. and Pentamer
Kuby Figure 14-12
-Most abundant in secondary responses -Crosses placenta (FcRn) -Complement activation -Binds to FcR in phagocytes - 4 Subclasses - 150,000
Figure 3.15a
8 Crosses placenta Crosses placenta Crosses placenta Complement Activator Complement Activator Complement Activator Fc binding Fc binding -Complement activation -First Ab produced in neonate -First antibody produced after challenge - Mucosal transport (to some degree) - Monomer on B cells - J chain: polymeric - 900,000
1
2
- Dimer in mucosal secretions - Mucosal transport - Monomer in circulation Secretory Component - J chain (polymeric) and Secretory components - 150,000 – 600,000
Role of IgE in allergic reactions IgD - IgE antibodies mediate the immediate- hypersensitivity (allergic) reactions. - Role unknown - IgE binds to Fc receptors on the - Present on the membranes of blood basophils and surface of tissue mast cells . MATURE Cross-linkage of receptor-bound IgE B cells Marker!! molecules by antigen (allergen) - 150,000 induces degranulation of basophils and mast cells.
A variety of pharmacologically active mediators present in the granules are released, giving rise to allergic manifestations
190,000
9 SUMMARY Antigenic Determinants on - IgA and IgM are secreted across epithelial surfaces Immunoglobulins - IgG, IgD and IgE can be found only within the • Abs are glycoproteins and themselves very body - in serum or lymph . immunogenic - IgA and IgM are also found in serum and lymph BUT • Epitopes on immunoglobulins are divided IN ADDITION can also be found in secretions such as mucous secretions, saliva and tears. into: – ISOTYPIC - The IgA and IgM found in external secretions differs from that found in serum by the presence of an – ALLOTYPIC additional component referred to as the "secretory – IDIOTYPIC component".
- This component is acquired as the IgA or IgM is transported across the epithelial cell barrier.
**
Constant region determinants that define each antibody Allelic variation (Allotypes): IgG of a particular class may be class and subclass slightly different between individuals ( e.g. variation in the IgG amino acid sequence) The function of antibody varies depending on which heavy chain Note: This type of variation has no effect on antibody is used. function.
RECAP - Sequence variation in antibodies:
1. Different light changes - no significant functional effect
2. Different heavy chains - very significant functional effect - isotypic variation
3. Allelic variation between individuals - no large functional effect - allotypic variation Generated by variation in amino acid sequence in the V H and VL. Most exactly, in the CDRs in the V regions 4. Variation in the antigen-binding site - idiotypic variation Variation in the antigen binding site (Idiotypes )
Remember: Idiotype = Ag binding site
10 B Cell Receptor (BCR): -Short cytoplasmic tail (3- Ig Superfamily 28 aa) ….signaling? • Divergence from a common gene ancestor coding -Signaling through a for 110 aa. heterdimer, Ig-α and Ig-β • A member MUST have a “typical” Ig domain or - Ig molecule + Ig-α/Ig-β is fold 110 aa with an intra chain disulfide bond the BCR 50-70 aa apart.
- The heterodimer molecule • Most members do not bind Ag!! Then, they must is member of the Ig facilitate interaction with surface proteins superfamily group • You must know members with roles in: a) immune function, b) Receptor/Signal transduction, and c) Adhesion
Receptors Immune Function
Neonatal
Monoclonal Antibodies
• Kohler & Milstein 1975 • Fusion of normal, activated B cell and plasmacytoma (cancerous plasma cell) • Hybrid: immortal, secrete Ab, hypoxanthine
11 1 Plasmacytoma VS B cell 2 3 • Plasmacytoma: 4 – Cancerous plasma cell (Immortal) – Does not secrete Abs – Lacks HGPRT 5 • Normal spleen B cell – Limited life span – Secretes Abs – Possess HGPRT
RESULTS: Applications
Spleen B cell Hybrid Plasmacytoma **
Die in culture Immortal, Secretes Lacks HGPRT Ab, Possess hypoxanthine
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